| Title:
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AAV Delivery of GRP78 Promotes Human RPE Cell Survival under Tunicamycin-induced ER Stress
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| Author(s):
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Shima Ghaderi, Zahra-Soheila Soheili, Shahin Ahmadian, Hamid Ahmadieh, Shahram Samiei
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| Presentation Type:
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Oral
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| Subject:
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Retina and Retinal Cell Biology
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| Others:
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Presenting Author:
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| Name:
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Shima Ghaderi
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| Affiliation :(optional)
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Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
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| E mail:
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shima.ghaderi@gmail.com
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| Phone:
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02633552887
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Mobile: |
09124634506
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Abstract (Max 200 words)
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| Purpose:
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Adeno associated virus (AAV)-mediated gene delivery of 78kDa glucose-regulated protein (GRP78) attenuates the condition of endoplasmic reticulum (ER) stress and prevents apoptotic loss of photoreceptors in retinitis pigmentosa (RP) rats. In the current study we overexpressed Grp78 with the help of AAV2 in human retinal pigmented epithelium (hRPE) cell cultures and examined its effect on cell adaptive response to ER stress. Although RPE degeneration is an integral part of RP, it seems a disregarded issue in investigations of retinal degenerative diseases. Hence the purpose of this work was studying potential stimulating effect of GRP78 on survival of ER stressed hRPE cells, as an in vitro model for RPE degenerative diseases.
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| Methods:
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hRPE cells were isolated from neonatal human cadaver eye globes, cultured in culture medium and characterized using immunocytochemistry. Recombinant rAAV/Grp78 viral vector was produced by triple-transfection of HEK293T packaging cells using AAV Helper Free System kit. To measure overexpression of Grp78, RPE cells were transduced by recombinant virus, and Grp78 expression was assessed by real-time PCR and western blotting. To investigate the effect of Grp78 overexpression on unfolded protein response (UPR) markers under ER stress, RPE cultures were transduced by recombinant virus and treated with ER stressor drug, Tunicamycin. Expression of four UPR markers were assessed by real-time PCR and western blotting.
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| Results:
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Our performed experiments found that GRP78 elevation in hRPE cells affects adaptive phase of UPR signaling network, through enhancement of UPR master regulators [PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6)] and decrease of pro-apoptotic downstream outputs [growth arrest DNA-damage-inducible gene 153 (GADD153)]. We detected 7.2-fold increase of PERK, 2.8-fold increase of ATF6 and 50% decrease of GADD153 in unstressed cells, in addition to 3-fold up-regulation of PERK, 5.4-fold up-regulation of ATF6 and 3-fold down-regulation of GADD153 at presence of drug-induced ER stress.
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| Conclusion:
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Together these findings demonstrate that elevated levels of GRP78 reveals promoting effect on ER-stressed hRPE survival, through anti-apoptotic actions in the early UPR. This survival factor can be recruited as a useful therapeutic target for future treatments of RPE degenerative diseases.
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| Attachment:
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